calcium phosphate—dna nanocomposites: morphological studies and their bile duct infusion for...

Calcium Phosphate—DNA Nanocomposites: MorphologicalStudies and Their Bile Duct Infusion for Liver-DirectedGene Therapy

Ruchi Singh, Amit Saxena, and Subho Mozumdar*

Department of Chemistry, University of Delhi, Delhi, India

In this study, liver-directed gene transfer in rats with calcium phosphate (CaP) nanoparticles and the effect of the route ofadministration and surgical manipulations on transfection efficiency is reported. Formulations of CaP nanoparticles entrap-ping plasmid DNA (pDNA) were prepared by the reverse micellar method using two different surfactants. Transmissionelectron microscopy, scanning electron microscopy and dynamic light scattering were used to characterize the CaP–DNAnanocomposites. The morphological characteristics of the formulations showed a strong dependency on temperature. Gelelectrophoresis experiments indicated that there was no degradation of the encapsulated pDNA, and in vitro cell transfection inHEK-293 and primary hepatocytes from rats as well as in vivo intraductal delivery experiments suggested that CaP nano-particles led to significant and prolonged transgene expression. Therefore, our methodology gives a stable and viable formu-lation for hepatic gene therapy. Low-DNA dosage entrapped in CaP nanoparticles makes it an effective gene delivery systemfor clinical applications.

Introduction

Gene therapy presents a promising and novel ap-proach for the treatment of a wide variety of humanailments.1–4 One of the major requirements for genetherapy is the effective transport of DNA through thecell membrane, although the processes through whichthe transfer occurs are not clearly defined.5,6 Vectors forgene transfer can be broadly categorized as viral and

nonviral. Although synthetic gene delivery systems offerseveral advantages over viral vectors in terms of the easeof production and reduced risk of cytotoxicity andimmunogenicity,7,8 their use has been limited by therelatively low transfection efficiency. Traditional nonvi-ral vectors involve condensation of the DNA with mul-tivalent cations such as polyamines, positively chargedpolymers, and peptides. However, these carriers do notlead to a satisfactory amount of transgene expression.This could be attributed to the low endosomal escape,less protection of DNA from nuclease degradation, andinefficient nuclear uptake.9 Developing trends involvethe use of inorganic nanocomposites, which are largelyunexplored synthetic systems. Some of the novel inor-ganic nonviral vectors for gene delivery that have been

Int. J. Appl. Ceram. Technol., 5 [1] 1–10 (2008)

Ceramic Product Development and Commercialization

Dr. Subho Mozumdar and Mr. Amit Saxena acknowledge the financial support of D.S.T.

in the form of research grants (SR/S1/PC-03/02 and SR/S5/NM-63/2002). Ms. Ruchi

Singh thanks the Lady Ratan Tata Foundation for providing her with Research Fellowships.

*[email protected]

r 2008 The American Ceramic Society

reported in recent years are silica nanoparticles,10 inor-ganic nanorods,11 nanotubes,12 layered double hydrox-ides (LDH),13 and other inorganic phosphatenanoparticles.14 CaP coprecipitation is one of themost common in vitro methods for DNA transfer intocell lines. This method is based on the fact that divalentmetal cations, such as Ca21, Mg21, Mn21, and Ba21,can form ionic complexes with the helical phosphates ofDNA15 and may provide a stabilizing function to cer-tain DNA structures.

We have previously reported the use of plasmidDNA (pDNA) encapsulated in CaP nanoparticles asgene delivery carriers and have specifically targeted theseparticles to liver cells after appropriate surface modifi-cation.16 CaP nanoparticles, as a component of nonviralvectors, promote the delivery of DNA from the cyto-plasm into the nucleus. It has been proposed that cal-cium ions form constituents of body fluids and arehighly biocompatible in trace amounts, as required forgene delivery.

The anatomy and physiology of the liver shows thatthe organ possesses various characteristics that make itvery attractive for gene medicine applications. First, thehepatic endothelium possesses fenestrae of 100 nm indiameter, which occupy about 6–8% of the sinusoidalsurface area. These sinusoidal and bile canaliculus fe-nestrae can permit easy and direct transport of macro-molecules and nanoparticles to hepatocytes.17 Second,there are many metabolic diseases that result from a de-fect or deficiency of hepatocyte-derived gene products.Finally, the liver is a highly vascular organ and thereforethe products of the transgene expression in this organcan easily access systemic circulation.

A number of delivery systems have been developedto deliver genes to the liver. Galactosylated cationicliposomes, galactosylated poly (l-lysine), and gala-ctosylated polyethylenimine (PEI) have been synthe-sized and evaluated as liver-targeted gene carriers,because the galactose moiety can be specifically recognizedby asialoglycoprotein receptors on hepatocytes.18–20

Moreover, the optimization of administrationroutes can also play a crucial role in liver-directedgene delivery. Infusion through the portal vein andbile duct has been shown to enhance gene transfectionin the liver.21–24 In particular, the bile duct route pro-vides many advantages over systemic administrations. Itdirectly delivers complexes to the liver parenchymalcells, bypassing the Kupffer cells. This not only im-proves the delivery efficiency to the hepatocytes, but also

avoids the opsonization of particles in serum. This cantherefore lead to directed gene expression in the he-patocytes. Furthermore, bile duct infusion can beachieved in the clinical setting through routine end-oscopic retrograde cholangiopancreatography.

In this study we examined whether a liver targeted,potential gene-transfer system could be achievedthrough bile duct infusion of CaP nanoparticles usinga very low dose of DNA. The term nanocomposites isused because we obtain different morphologies of thesamples under different conditions of preparations. Onthe one hand, we obtain regular nanoparticles when theparticles are prepared under room temperature condi-tions as reported in our earlier publication16 and nano-rings (as shown in Fig. 2 of the manuscript) when thepreparations are filtered and stored at �201C.

Experimental Procedure

Materials

The surfactants Aerosol OT (AOT), sodium bis(2-ethylhexyl)sulfosuccinate, and TritonX-100, of analyti-cal grade, were purchased from Sigma (St. Louis, MO).The plasmid encoding the 6.4 kb firefly luciferase(pCMV-luciferase VR1255_C) driven by the cytome-galovirus (CMV) promoter/enhancer (luciferase plas-mid) was extracted and purified in the laboratoryusing QIAGEN columns. p43-Clz1 pDNA (7.8 kb) en-coding the b-galactosidase gene driven by the CMVpromoter was purchased from Aldevron LLC (Fargo,ND). n-hexane (AR grade) was purchased from ThomasBaker. Cell culture media (DMEM) was obtained fromGibco. Fetal calf serum (FCS) and penicillin–strepto-mycin solution were purchased from Sigma-Aldrich.Branched PEI (average molecular weight 25 kDa) waspurchased from Sigma. PEI was purified by dialysisagainst water (dialysis tubing with MWCO 3500;Pierce, Rockford, IL).

Methods

Preparation of CaP–DNA Nanoparticles: CaP nanopar-ticles were prepared by modifying our previously re-ported method.16 In 25 mL of 0.1 M solution of AOTin hexane (or 0.1 M solution of TritonX-100 in cyclo-hexane), 70 mL aqueous solution of inorganic salt(20%w/v CaCl2) and an aqueous solution of 2.94 mgof pDNA were dissolved by continuous stirring for 12 h

2 International Journal of Applied Ceramic Technology—Singh, Saxena, and Mozumdar Vol. 5, No. 1, 2008

to form microemulsion A. In another 25 mL of a 0.1 Msolution of AOT(or TritonX-100 solution), 70 mL ofNa2HPO4 (5% w/v) and an aqueous solution of 2.94 mgof pDNA were dissolved by continuous stirring for 12 hto form microemulsion B. The w0 values, the molar ra-tio of water to surfactant in all the microemulsions wereadjusted to 10 by making up the total aqueous volumeto 450 mL. Both microemulsions were optically clearsolutions in the case of AOT. However, in the case ofTritonX-100, n-hexanol was added as a cosurfactant toobtain an optically clear solution. Microemulsion B wasthen slowly added to microemulsion A at a rate of about4 mL/ h with continuous stirring at 41C. The resultingsolution was then further stirred for 12 h in the abovecold condition. Development of translucency indicatedthe formation of nanoparticles in the aqueous core ofthe reverse micellar droplets. Hexane (or cyclohexane)was evaporated off and the cake was dissolved in 10 mLof ethanol. Centrifugation was performed at 4000 rpmfor 20 min to extract the nanoparticles. Pelleted nano-particles were washed thrice with ethanol to remove ex-cess AOT. The nanoparticles were redispersed in 10 mLof 1� phosphate-buffered saline (PBS). Finally, 100 mLof FCS was added to the dispersed nanoparticles to ob-tain a fine dispersion.

Serum Stability of CaP–DNA Nanoparticles: pDNA-loaded CaP nanoparticles were dispersed in 10% serumof 500 mL volume and incubated at 371C in a shakerincubator. Twenty microliters of the samples was re-moved at specific intervals of time (0, 0.1, 0.5, 1, 2, 4, 6,8, and 12 h) and stored at �201C. Subsequently, thesesamples were subjected to electrophoresis on a 0.8%agarose gel.

In Vitro Transfection: HEK-293 cells were grown inDMEM containing 10% FCS under standard condi-tions for 24 h till 80% confluency was reached. Afterconfirmation of cell viability using trypan blue exclu-sion, 12-well plates were seeded with 8� 105 cells perwell and 1 mg of DNA encapsulated in nanoparticles wasadded in each well. PEI–DNA complexes prepared at anN/P ratio of 10 were used as a positive control. After4 h, the transfection medium was removed and the cellswere washed. After 2 days of further incubation in se-rum-containing media, the wells were washed with PBS.Cells were lysed with a lysis buffer (Promega). The lu-ciferase activity in cell extracts was measured on aluminometer (LUMAT LB 9507, Berthold, Germany).The relative light units (RLU) were normalized against

protein concentration in the cell extracts (BCA proteinassay kit, Pierce).

Isolated hepatocytes were seeded 24 h beforetransfection into six-well collagen-coated plates at aninitial density of 8� 105 cells per well. Before transfect-ion, the medium in each well was replaced with 2 mL offresh William’s E medium. Nanoparticles containing2 mg of DNA were added to each well. After incubationfor 4 h, the medium was replaced with 2 mL of freshWilliam’s E medium. Forty-eight hours later, cells werelysed with cell lysis buffer (Promega). The luciferase ac-tivity in cell extracts was measured as described above.

Bile Duct Infusion of CaP–DNA Nanoparticles: Six to 8-week-old male Wistar rats were obtained and housed inthe National University of Singapore’s Animal HoldingUnit. Rats were maintained on ad libitum rodent feedand water at room temperature with 40% humidity. Allthe animal procedures were the ones approved by theNational University of Singapore, Faculty of MedicineAnimal Care and Use Committee. Wistar rats (male,200–250 g, 12–16 per group) were used for the exper-iments. The animals were laparotomized under generalanesthesia and the liver was then surgically isolatedfrom the surrounding tissue. A 33 G needle was insert-ed into the common bile duct and a tie was used tosecure the needle. Nanoparticles and naked DNA wereadministered at the dose equivalent 2 mg of plasmid in4 mL of medium into the common bile duct over a20-min period using a syringe pump and a 33 G needle.A tie was then placed around the bile duct between theliver and the point of infusion to prevent back flow,and the needle was withdrawn. After 30 min, all tieswere removed.

On days 1 and 7, three rats from each group weresacrificed and the major organs (liver, heart, lung, spleen,and kidney) were harvested and stored at �801C for fu-ture analysis. Each liver was divided into four sectionscomposed of median, left, right, and caudate lobes. Twomililiters of lysis buffer (0.1% TritonX-100, 2 mM/Lethylenediaminetetraacetic acid, and 0.1 mol/L Tris-HClpH 7.8) per organ gram weight was used on each sample.The other major organs were homogenized and thensubjected to two cycles of freeze-thawing, followed bycentrifugation at 14,000 rpm for 10 min. The proteinconcentration in the supernatant of the samples wasdetermined by a protein assay kit (Pierce).

To determine luciferase activity, the luminescence of10 mL of supernatant was measured in a luminometer for

www.ceramics.org/ACT CaP–DNA Nanocomposites 3

10 s and the result was presented as relative light units pergram weight of tissue. The mean of the luciferase activityof the liver was the sum of the relative light units pergram weight of each lobe times the weight percentage ofeach lobe relative to the total liver weight.

Hepatic Toxicity: Blood samples were drawn on days1, 2, 3, and 7 for liver toxicity and liver function an-alyses (alanine transaminase (ALT) and aspartate trans-aminase (AST) activities, bilirubin). Hepatic toxicityrelated to the CaP nanoparticles and gene transferwere assessed by serial assays of serum aspartate ami-notransferase activities from days 0 to 7 and by histo-logical examination of the livers on day 3 afterintraductal infusion. AST and ALT were assayed at thesame time points using a multiparametric automaticanalyzer.

Histological Examination of DNA Distribution After Intra-biliary Infusion of the Nanoparticles: Two slices of hepatictissue were obtained from the middle of the right laterallobe of each liver and fixed for 2–3 days with neutral-buffered formalin (Baxter Scientific Products, McGawPark, IL). After fixation, the specimens were routinelyprocessed and embedded in paraffin. Paraffin sections(4 mm) were used for histopathological analysis (hem-atoxylin–eosin staining).

Detection of b-Galactosidase Gene Expression on FrozenSections of the Liver: Four milliliters of PBS or nano-particles/p43-Clz1 pDNA (200 mg) complexes were ad-ministered through the bile duct as described above.Two days after infusion, the rat livers were harvestedand frozen in liquid nitrogen. Five to eight micrometercryostat sections were obtained from different lobes ofthe liver. The sections were then fixed in 0.5% glut-araldehyde in PBS at 41C for 15 min and washed twicewith PBS. The sections were stained with a LacZReporter Assay Kit (tissue staining) (Invivogen, SanDiego, CA) at 371C for 2 h. The stained sections werewashed twice with PBS, counterstained with 0.1% (w/v)Nuclear Fast Red for 1 min, dehydrated, and mounted.

Statistical Analysis: Data were expressed as means1SD, and the statistical significance of differencesamong groups was assessed by Student’s t test as ap-propriate. P values o.05 were regarded as statisticallysignificant.

Results

Physical Characterization of the CaP–DNANanoparticles

pDNA encapsulated nanoparticles were formed inthe aqueous core of the microemulsion of AOT in hex-ane (or TritonX-100 in cyclohexane). The strategy in-volved the coprecipitation of CaP and DNA in theaqueous core of the reverse micellar droplets. A 1% se-rum solution of 1� PBS was used to obtain a fine dis-persion of nanoparticles. The size distribution of thenanoparticles showed that the particles were about50 nm, similar to the values we have reported earlier.16

In the case of nanoparticles prepared in TritonX-100 incyclohexane, a rapid change in morphological charac-teristics was observed when the formulations were storedat �201C. When the particles were filtered through0.2mm filters, a ring-like morphology of the particlescould be observed in about 30 min to 1 h storage at�201C. This is clear from the scanning electron micros-copy (SEM) image of the formulations (Fig. 1A, C, E,and G). However, the nonfiltered preparations did notshow such changes and were mostly of long filamentoustype (Fig. 1B, D, F, and H). What has been observedthrough the SEM study is that the size (aspect ratio) ofthe particles did not change considerably over time. Itremained almost close to an aspect ratio of 13 to 14.Dynamic light-scattering data were not very informativeas the particles were filamentous and not spherical.

The percentage of loading in a typical set turns outto be approximately 0.076% (this has been calculatedon the basis of the amount of DNA to the amount ofCaP).

The DNA-loaded CaP nanoparticles were redis-persed in 10% serum and their chemical and morpho-logical stability was examined. It was found that theparticles are quite stable as no released and degradedDNA bands could be observed in electrophoresis up to12 h (Fig. 2). DNA could be released from the CaPnanoparticles only under acidic pH.

In Vitro Transfection Study

The transfection efficiency against the HEK-293cells was comparable to that of PEI at the same DNAdose (Fig. 3). The cytotoxicity of the CaP nanoparticles,however, was much lower than that for PEI.


A. Filtered calcium phosphate-DNA B. Non-filtered calcium phosphate-DNA complex at 0 hour complex at 0 hour

C. Filtered calcium-phosphate -DNA D. Non-filtered calcium phosphate-DNA complex after 10 mints of freezing at complex after 10 mints of freezing

– 20 deg-centigrade at –20 deg-centigrade

E. Filtered calcium-phosphate –DNA F. Non-filtered calcium phosphate- DNA complex after 30 mints of freezing at complex after 30 mints of freezing

– 20 deg-centigrade at –20 deg-centigrade

G. Filtered calcium phosphate-DNA H. Calcium phosphate-DNA nano-rings complex after 1 hr of freezing at – 20 deg-centigrade

Fig. 1. Scanning electron microscopy images of calcium phosphate (CaP)–DNA nanoring formation. (A) Filtered CaP–DNA complex at 0 h,(B) nonfiltered CaP–DNA complex at 0 h, (C) filtered CaP–DNA complex after 10 min of freezing at �20 1C, (D) nonfiltered CaP–DNAcomplex after 10 min of freezing at �20 1C, (E) filtered CaP–DNA complex after 30 min of freezing at �20 1C, (F) nonfiltered CaP–DNAcomplex after 30 min of freezing at �20 1C, (G) filtered CaP–DNA complex after 1 h of freezing at �20 1C, and (H) CaP–DNA nanorings.


Because our major aim was to target liver cells, thetransfection ability of CaP nanoparticles was evaluatedagainst primary rat hepatocytes. As naked DNA canefficiently transfect the liver via hydrodynamic injection,it was added as a control in this cell culture experiment.The CaP nanoparticles produced a transfection levelthat was significantly more than naked DNA (Fig. 4). Apossible mechanism of transfection by the CaP–DNAnanoparticles has been put forward by Bisht et al.25

In Vivo Studies

Previous data have demonstrated that liver-directedgene transfer can be achieved by bile duct infusion.Compared with the portal vein and tail vein adminis-tration route, this may be a better choice for efficient

liver-directed gene transfer. In the time course study oftransgene expression after bile duct infusion using CaP–DNA nanoparticles and naked DNA, the former pro-duced a 30–40-fold higher luciferase expression in liverthan PBS or naı̈ve controls (Po0.01). The gene expres-sion level decreased to about 10-fold higher than thenaı̈ve control on day 7 (Fig. 5). The naked DNA-mediated transgene expression kinetics was different.On day 1, the gene expression level was about 17-foldhigher than CaP, but it decreased to the background onday 7. The more prolonged gene expression of the CaP

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Fig. 4. Transfection efficiency of calcium phosphate nanoparticlesin primary hepatocytes of the rat.

lane1: Standard plasmid DNAlane2: Cap nps at 0hr lane3: Cap nps after 0.1hr lane4: Cap nps after 0.5 hr lane5: Cap nps after 1hr lane6: Cap nps after 2hrs lane7: Cap nps after 4 hrs lane8: Cap nps after 6 hrs lane9: Cap nps after 8 hrs lane10: Cap nps after 12 hrs lane11: Standard plasmid DNA lane12: Molecular weight marker.

Fig. 2. Serum stability of the plasmid DNA (pDNA)-encapsulated calcium phosphate (CaP) nanoparticles at specific intervals of time. Lane1: standard pDNA (pDNA); lane 2: CaP nanoparticles at 0 h; lane 3: CaP nanoparticles after 0.1 h; lane 4: CaP nanoparticles after 0.5 h;lane 5: CaP nanoparticles after 1 h; lane 6: CaP nanoparticles after 2 h; lane 7: CaP nanoparticles after 4 h; lane 8: CaP nanoparticles after6 h; lane 9: CaP nanoparticles after 8 h; lane 10: CaP nanoparticles after 12 h; lane 11: standard pDNA; lane 12: molecular weight marker.


group was also supported by the b-galactosidase stainingin the liver sections (Fig. 6).

The acute hepatic toxicity related to the bile ductinfusion of CaP–DNA nanoparticles was assessed byanalyzing the serum AST andALT activities during theexperimental period and compared with the infusion ofnaked DNA. A slight increase in serum AST and ALTactivities was observed 1 day after the administration ofnaked DNA, followed by a rapid decrease to the normallevel by day 3 (Fig. 7). CaP–DNA nanoparticles in-duced a moderate increase of both ALT and AST ac-tivities, which were slightly higher than those of thenaked DNA group. It suggested that the hepatocellularinjury was transient. Throughout the experimental pe-riod, bilirubin, g-glutamyltransferases (g-GGTs), andcreatinine levels for all the groups were in the normalrange (5–30 IU/L), indicating that there was no ob-structive jaundice caused by the injection procedure.Our data have indicated that by day 4 after injection, alltransaminase levels returned to normal (Fig. 7).

Discussion

The difficult task of defining conditions for effec-tive in vivo gene delivery with nonviral DNA vectorsremains unresolved, with no ideal vectors currentlyavailable. Although various nonviral approaches have

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Fig. 5. Transgene expression following calcium phosphate–DNAnanoparticle and plasmid DNA delivery to the liver after day 7.

Fig. 6. (A) Frozen section of normal liver treated with naked DNA. (B) b-Galactosidase gene expression in the frozen section of liver afterbile duct infusion of plasmid DNA-encapsulated calcium phosphate nanoparticles.

AST: Aspartate transaminase

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Fig. 7. Serum alanine transaminase (ALT) and aspartatetransaminase (AST) levels at several time points followingintraductal delivery of calcium phosphate–LacZ nanoparticles.


been developed, any method needs to be optimized interms of the target disease and the transgene product.The most important information required is (i) targetcell-specificity of gene transfer, (ii) efficiency, (iii) du-ration of transgene expression, and (iv) the number oftransfected cells following in vivo application of a vec-tor. These characteristics are determined by the proper-ties of the vector used, as well as the route of itsadministration, biodistribution, interaction with biolog-ical components, and the nature of the target cells. Cell-specific gene transfer can be achieved by controlling thetissue disposition of pDNA, although the interaction ofthe pDNA complex with biological components mightlimit the specificity. Various approaches have been re-ported to increase the efficiency of transgene expression,from cationic lipids/polymers to physical stimuli, butnot all of those are effective under in vivo conditions.

We have chosen to pursue nonviral gene delivery viathe regional infusion of vector/DNA complexes, ratherthan attempting vector targeting following simple sys-temic or portal intravenous injection. Compared with theportal vein and tail vein administration route, this may bea better choice for efficient liver-directed gene transfer.Regional administration has many practical and theoret-ical advantages, as outlined in ‘‘Introduction’’ and couldwell be the method of choice in spite of the greater initialtime and effort required in each patient. With some or-gans, regional administration is not a substantial practicalproblem. With the liver, for example, canulation of thebile duct is a routine clinical procedure, performed underlight sedation with a duodenoscope.

A successful liver-targeted gene delivery will havewide implications for treating inherited and metabolicliver diseases, such as liver cancer and viral hepatitis(e.g., interferon a and g delivery), and systemic diseases,such as hemophilia A and B (factor VIII and IX deliv-ery). Successful in vivo gene therapy requires the devel-opment of a rational gene transfer approach that fulfillsvarious requirements for each target disease. Develop-ment of an efficient method of gene introduction totarget cells is the key issue in treating genetic and ac-quired diseases by in vivo gene therapy.

As far as the design of the gene delivery vector isconcerned, inorganic nanoparticles entrapping biomol-ecules clearly display a wide range of diversity and po-tential applications.26 Their primary advantage stemsfrom the fact that they are not subjected to microbialattack and exhibit excellent storage stability. Moreover,they can be prepared at a low temperature and are rel-

atively inexpensive. Some ceramic particles of nano-dimensions have been prepared encapsulating or inconjugation with DNA, with the purpose of using asnonviral vectors as well as serving as adjuvants forvaccines and other applications.27–30 It is known thatCa21, Mg21, Mn21, and Ba21 can form ionic com-plexes with biological macromolecules like DNA, andthese complexes can have easy transportability across thecell membrane via ion channel-mediated endocytosis.31–33

Furthermore, these divalent cations form constituentsof body fluids and are highly biocompatible in traceamounts as required for gene delivery applications.Among these inorganic compounds, the most common-ly used salt is CaP, whose microparticles and nanopar-ticles have been developed as a delivery system as well asan adjuvant for DNA vaccines.34,35 Although the use ofprecipitated CaP for in vitro transfection is a routinelaboratory procedure,36 the method is hampered by thedifficulty of applying it to in vivo studies, especially de-livery of DNA to any particular cell type. Because ofbulk precipitation of CaP, the method also suffers fromvariation in CaP–DNA particle size that causes varia-tions among experiments.30 Our method of preparingCaP–DNA nanocomposites, with uniform size distri-bution, by the simple reverse method is a clear step for-ward in this direction.

The ultimate goal of using carriers for gene therapyis to protect and transport the genetic material into thecell and finally into the nuclear compartment, avoidingintracellular degradation. Use of CaP alone or in com-bination with other vectors exerts its positive effect onthe gene transfer by stimulating cellular uptake of DNAin a process involving endocytosis of the membrane-bound DNA complex.8–10 CaP nanoparticles encapsu-lating DNA were added to HEK-293 cells to test thetransfection efficiency. The transfection efficiency ob-tained by these nanoparticles is quite significant. ThePEI–DNA complex was used as a positive control. Hightransfection efficiency of CaP nanoparticles can be at-tributed to the ultralow size of the nanoparticles com-bined with the effect of calcium ion-mediatedendocytosis. The bile duct offers a relatively easy, rou-tine, and noninvasive approach for gene delivery to theliver. The particular clinical applications made possibleby this approach will depend on the precise localizationof gene expression (e.g., hepatocytes, biliary epithelium)and the efficiency of gene transfer (in terms of the pro-portion of cells expressing the therapeutic gene), as wellas the longevity of gene expression.


To understand the transfection mechanism in livercells, an experiment was undertaken in primary he-patocytes of the rat. This experiment was carried outto support the in vivo studies for liver-targeted gene de-livery through CaP nanoparticles. Parenchymal livercells are useful target cells for gene delivery becausethey can perform a host of posttranslational modifica-tions that may be required for the activity of certaingene products. The cells are highly active metabolically.Moreover, the liver has a rich blood supply that can beuseful for the delivery of genes to the liver. The dataconfirm that the transfection efficiency is highly signifi-cant in hepatocytes.

In terms of optimizing gene transfer efficiencymediated by nonviral vector, most attention has beenfocused on the design and modification of the gene car-riers and gene constructs. However, increasing evidencehas suggested that the optimal characteristics of a genecarrier and the construct are also dependent on the tar-get tissue and cells and, equally important, the admin-istration route. The same plasmid and gene carrier givenby two different administration routes can result in verydifferent levels of gene expression. Hence, the optimi-zation of gene vector/construct and administration routeshould go hand in hand.

Finally, the duration of transgene expression is acomplex function involving variables such as cell type,transfection method, and plasmid construct. Immuneresponse often reduces the level and duration of trans-gene expression. In addition, the number of cellstransfected is important, especially in cases in whichtherapeutic protein localizes within target cells. Success-ful clinical application of nonviral gene delivery meth-ods relies on the development of such methodsoptimized for a particular target disease.

Acknowledgments

The authors deeply thank Prof. Kam Leong of theJohns Hopkins University for extending his facilities atSingapore and Dr. Dai Hui for helping with the surgicalprocedures and animal experiments.

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